**6.5. CYP1A1 and CYP2B in different species after treatment with BPA**

In Table 5A-B we reported the results of the mean levels of immunofluorescence of CYP1A1 (A) and CYP2B (B), revealed in cultured fibroblasts of killer whale (MOO12) treated with BPA, expressed as index numbers.


**Table 5.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with BPA. The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of these proteins. D.C. = death cells.

Only one species, the killer whale, was treated with this potent estrogenic compound since the study to evaluate the cetacean susceptibility to BPA has just started in our laboratories. However, this preliminary experiment confirms that BPA is a potent CYP2B inhibitor [35]

while it gives information on CYP1A1 inductive capacity (Figure 6 A-B). To be highlighted that the highest used dose (100 μg/ml) is 100% lethal for fibroblast cells.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 63

**7. Qualitative and quantitative mica protein expression as toxicological** 

Cetacean morbilliviruses and papillomaviruses as well as *Brucella* spp. and *Toxoplasma gondii* are thought to reduce population abundance by inducing high mortalities, lowering reproductive success or by synergistically increasing the virulence of other diseases. Severe cases of lobomycosis and lobomycosis-like disease (LLD) may contribute to the death of some dolphins [37]. Environmental contamination seems to play a role in these diseases because many pollutants are known immunosuppressants and can markedly affect the immune status of the cetacean specimens [38, 39, 40, 41, 42]. As already mentioned, the cetacean skin is an important tissue of the immune system and the MICA protein, used in this study as toxicological stress marker of the immune system, is expressed in fibroblasts. In fact, the aim of this study was to evaluate the MICA protein expression in fibroblast cell cultures of cetaceans (skin biopsies of free-ranging animals and skin samples of stranded cetaceans dead within 2-12 h). Here we present the immunofluorescence technique in cultured fibroblasts used for qualitative and quantitative evaluation of MICA expression, induced by treatment with OC mixture, flame retardants, PAHs and BPA, as toxicological stress marker of the immune system of different species of odontocetes (sperm whale (PMAS1)*,* killer whale (OO12), striped dolphin (RT23), long-beaked common dolphin

The basal level of MICA, evaluated with immunofluorescence technique in the fibroblasts of different cetacean species before treatment with different mixtures, is the first important result of this research step as it provides an indication of the immune status of these marine mammals. The results, expressed as immunofluorescence for cell (AUF/nucleus) mean

**Bryde's whale (MBE3)** 50482.0

**Killer whale (MOO12)** 195378.0 **Sperm whale (PMAS1)** 12647.0 **Bottlenose dolphin (TTA1)** 31473.0 **Striped dolphin (RT23)** 18511.0 **Fin whale (RT25)** 31325.0

**Long-beaked common dolphin (MDC12)** 94751.0

**Table 6.** Basal levels of immunofluorescence (AUF/nucleus) of MICA in fibroblast cells of Bryde's whale, long-beaked common dolphin, killer whale, sperm whale, bottlenose dolphin, striped dolphin

We can highlight that the three specimens belonging to the three species sampled in the Sea of Cortez (Bryde's whale, long-beaked common dolphin and killer whale) showed higher basal activity of MICA with respect to all Mediterranean specimens, regardless of the

*MICA (UAF/nucleus)* 

(MDC12)) and mysticetes (fin whale (RT25)*,* Bryde's whale (MBE3)).

values are presented in the Table 6 and, as histograms, in the Figure 7.

**7.1. Basal levels of MICA in different species** 

and fin whale.

**stress marker of the immune system** 

**Figure 5.** A-E: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-E) in fibroblast cells of Bryde's whale (MBE3) (A), sperm whale (PM6) (B), Risso's dolphin (GGL1) (C), Striped dolphin (RT1) (D) and longbeaked common dolphin (MDC12) (E) treated with PAHs. DAPI and Alexa Fluor 594 (Intensity 200ms) images of Acetone and the three PAH treatments.

**Figure 6.** A-B: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-B) and CYP2B (C-D) in fibroblast cells of killer whale (MOO12) (A, B) treated with BPA. DAPI and Alexa Fluor 594 (Intensity 200ms) images of Ethanol and the four BPA treatments.
